Purpose :
A broad variety of progressive retinal degenerations are caused by inherited defects in P/rds, a tetraspanin protein essential for proper formation and stability of the outer segment (OS) organelles of rod and cone photoreceptors. A subset of pathogenic defects affect the protein's C-terminus; however, the normal function of this domain is not yet known, and hinders a detailed understanding of disease etiology. The current study investigates the importance of the P/rds C-terminus in vivo for OS structure/function and photoreceptor function and viability.

Methods :
P/rds knock-in mice were generated using the CRISPR/Cas9 system in conjunction with pronuclear microinjection into single-cell zygotes. Prph2 exon 3 was targeted for cleavage proximal to Tyr285, and a ssDNA donor template was utilized to introduce a C>A transversion, mimicking a putatively pathogenic mutation in human P/rds. Founder mice identified via DNA sequencing were backcrossed to create C57BL/6J congenic lines. Age-matched gene-edited mice and littermate controls were analyzed using histology, immunohistochemistry, Western blotting, transmission electron microscopy, and electroretinography. Phenotypic effects of the Tyr285stop mutation were validated using lines derived from independent founders.

Results :
Two independent lines of Tyr285stop P/rds mice have been generated; each has been transferred to the C57BL6/J background to eliminate potential off-target effects of gene-editing. Comparison of the two lines confirmed a consistent phenotype and demonstrates that a Tyr285stop nonsense mutation associated with progressive retinal degeneration in humans also causes retinal dystrophy in mice. This defect disrupts photoreceptor OS morphogenesis in a distinctive manner, impairs retinal function, and demonstrates an essential role for the P/rds C-terminal domain in photoreceptor structure/function and the maintenance of retinal health.

Conclusions :
This work presents the first animal model for Prph2-associated disease to investigate a defect in the protein’s cytoplasmic C-terminus. It also demonstrates the utility of CRISPR/Cas9 gene-editing for efficiently producing murine models for progressive retinal disease. The results to date support the presumed pathogenicity of a heterozygous Tyr285stop mutation in P/rds, and allow investigation of protein domain structure/function in vivo.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.